Tumor cells escape attack from the immune system through various mechanisms, including immune evasion and/or immune suppression. In order to achieve better therapeutic effects using immune modulatory therapy, it is important to study the mechanisms underlying tumor-induced immunosuppression of the tumor-specific T cell response. We found that the growth of various carcinomas induces a significant increase in the numbers of myeloid suppressor cells (MSC) in tumor infiltrating leukocytes (TILs), spleen, and bone marrow of tumor-bearing mice. We hypothesize that MSC suppress the tumor-specific T cell response locally and inactivate tumor-specific T cell response in vivo. The preliminary results support the hypotheses we proposed in our previous submission and indicate that sorted Gr-1+CD115+ MSC isolated from spleen and bone marrow of tumor-bearing mice drastically inhibit IFN-y-producing tumor antigen- activated T cells, significantly reduce expression of IL-2 by T cells, induce expression of IL-10, TGF-p, FoxpS, and Tob, and induce the development of T regulatory cells (Treg) that are anergic and suppressive. Clonal expansion of adoptively transferred tumor antigen-specific T cells occurred during an early stage of tumor growth, but expansion is suppressed by adaptively transferred MSC. At a later time point, tumor-bearing mice developed tolerance associated with FoxpS and Tob expression in the expanded tumor antigen-specific T cells. Furthermore, adoptive transfer of sorted Gr-1+CD115+ MSC, but not Gr-rCD115'or Gr-1+CD115" cells, can induce tumor specific anergy and Treg development in recipient tumor-bearing mice. These results provide strong evidence of an immunoregulatoryfunction for MSC in vivo in the establishment of tumor- specific tolerance and the development of Treg. To achieve persistent anti-tumor immunity and to improve the therapeutic effect of immunomodulatory treatments, tumor-induced immunosuppression must be overcome to allow for an effective T cell response. Three specific aims will be pursued to understand the mechanisms regulating MSC accumulation and the in vivo suppressive function of MSC in tumor bearing mice: 1) to study the mechanisms underlying the accumulation and differentiation of MSC in tumor-bearing mice; 2) to investigate the mechanisms of MSC-mediated tumor specific T cell inactivation in vitro and in vivo; and 3) to prevent the accumulation of MSC and to block their immune inactivation mechanisms, thereby improving immune modulated cancer therapy for treating advanced tumors. Successful completion of these studies will result in a better understanding of the mechanisms underlying tumor-mediated immune suppression by MSC. This information will be utilized as the scientific foundation for the development of a novel therapeutic modality that would counteract the immune suppression associated with advanced malignancy. The ablation of MSC-mediated suppression should significantly augment the efficacy of our immune activation protocol (IL-12 gene delivery plus 4-IBB activation) for patients with advanced cancer.